Peptide antibacterial compound as well as preparation method and application thereof

Abstract

The invention provides a peptide antibacterial compound as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The invention provides a peptide compound zhenxinmyxin as shown in a formula I. The peptide compound has good structural characteristics, the antibacterial effect on staphylococcus aureus can be remarkably improved, the minimum inhibitory concentration on staphylococcus aureus ATCC 25923 and USA300 is 4 [mu] g / mL, and the peptide compound can be used for preparing drugs for resisting gram-positive bacterial infection. The peptide compound is a novel peptide structure compound extracted from fermentation liquor of streptomyces albidoflavus NX-12, raw materials are easy to obtain, the preparation method is simple and convenient, and the peptide compound is suitable for large-scale popularization and application.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to a peptide antibacterial compound, a preparation method thereof, and an application thereof. Background Art

[0002] Peptide compounds are formed by amino acids linked through peptide bonds and belong to a class of natural or synthetic molecules. Due to their flexible structures and variable chemical configurations, peptide compounds have shown obvious advantages in numerous biological activity studies. According to their specific structures, such compounds can be classified into linear or cyclic peptides, and their three-dimensional conformational characteristics endow them with excellent target-binding selectivity and significant biological activities. Compared with traditional small molecule drugs, peptide compounds have lower toxicity and also exhibit good characteristics in pharmacokinetics, so they have attracted much attention in new drug research and development. Peptide compounds have a wide range of applications in clinical treatment, covering antibacterial, anti-tumor, antiviral, and immunomodulatory fields. In particular, there are many successful application examples in the antibacterial field. For example, polymyxins B and E (i.e., colistin) are important anti-Gram-negative bacteria drugs and are often used to treat severe infections caused by drug-resistant bacteria, such as infections caused by carbapenem-resistant Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus. Daptomycin, as a cyclic lipopeptide antibiotic, is specifically used to combat complex skin and soft tissue infections caused by drug-resistant Gram-positive bacteria [7]. These peptide antibiotics achieve their antibacterial effects through mechanisms such as interfering with bacterial cell membranes and inhibiting protein synthesis, showing significant advantages against drug-resistant strains.

[0003] However, with the widespread use and abuse of antibiotics, the problem of bacterial drug resistance has become a global public health challenge, and the efficacy of traditional antibiotics has decreased significantly. Therefore, there is an urgent need to develop new antibiotics, especially peptide antibacterial drugs with unique antibacterial mechanisms and broad-spectrum activities, to cope with the threat of multi-drug resistant pathogens.

[0004] Advances in genomics have brought important opportunities for the discovery and development of novel peptide compounds. Through the study of microbial genomes, scientists can identify and predict the synthetic gene clusters of peptide compounds. For example, through genomic mining technology, the daptomycin gene cluster in Streptomyces has been successfully discovered, enabling the large-scale production and widespread application of daptomycin in the treatment of drug-resistant Gram-positive bacterial infections, especially showing significant efficacy against multi-drug resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA). The application of gene mutation and synthetic biology techniques has also made it possible to modify the polymyxin gene cluster, thereby improving its production efficiency and antibacterial properties. Genomics methods not only accelerate the development of novel peptide antibacterial drugs but also provide rich resources for the innovation of peptide antibiotics. However, only natural L-type antibacterial peptides can be obtained through genetic engineering, and they are very sensitive to proteolysis, so their stability in vivo is very poor. Summary of the Invention

[0005] The object of the present invention is to provide a peptide antibacterial compound and its application.

[0006] To achieve the above object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides an antibacterial compound, characterized in that the structural formula of the antibacterial compound is shown in Formula I:

[0008]

[0009] The present invention also provides a medicinal salt formed by the compound.

[0010] The present invention also provides a preparation method of the peptide antibacterial compound, comprising the following steps:

[0011] S1. Coat the spores of Streptomyces albidoflavus NX-12 on a solid medium and culture at 26-28 °C until the spores cover the plane;

[0012] S2. Pick Streptomyces albidoflavus NX-12 into a liquid medium for fermentation culture;

[0013] S3. Use ethyl acetate to concentrate the fermentation product under reduced pressure to obtain a crude extract;

[0014] S4. Purify the extract obtained in S3 to obtain the antibacterial compound;

[0015] The Streptomyces albidoflavus NX-12 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 33132.

[0016] Preferably, the solid medium is MS medium; the liquid medium is G2 medium.

[0017] Preferably, the conditions for fermentation culture are: 26 - 30 °C, 200 - 240 rpm, and culture for 4 - 6 d.

[0018] The present invention also provides the application of the peptide compound in the preparation of a drug for inhibiting Staphylococcus aureus.

[0019] The present invention also provides a biological antibacterial agent, which uses the peptide antibacterial compound and / or the medicinal salt as the active ingredient, and also includes pharmaceutically acceptable excipients.

[0020] Preferably, the pharmaceutically acceptable excipients account for 0.1 - 99.9% of the total weight of the pharmaceutical composition by weight.

[0021] Preferably, the dosage forms of the biological antibacterial agent include tablets, capsules, liquid preparations, and dry suspensions.

[0022] Streptomyces alboflavus NX - 12 of the present invention is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 33132, the deposit date being December 20, 2024, and the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0023] By adopting the above technical solutions, the present invention has the following beneficial effects: The present invention provides a peptide compound zhengxinmyxin shown in Formula I. This peptide compound has good structural characteristics, can significantly improve the antibacterial effect against Staphylococcus aureus, and the minimum inhibitory concentration against Staphylococcus aureus ATCC 25923 and USA300 is 4 μg / mL, and it can be used to prepare drugs for treating Gram - positive bacterial infections. The peptide compound of the present invention is a compound with a new peptide structure extracted from the fermentation broth of a strain of Streptomyces alboflavus NX - 12. The raw materials are easily available, and the preparation method is simple, which is suitable for large - scale popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a flow chart for the extraction and separation of the peptide compound shown in Formula I.

[0025] Figure 2 It is the high - resolution electrospray mass spectrum of the compound shown in Formula I.

[0026] Figure 3 For the compound shown in Formula I 1 1H NMR spectrum (CD3COCD3, 600 MHz).

[0027] Figure 4 of the compound shown in Formula I 13 13C NMR spectrum (CD3COCD3, 150 MHz). Detailed implementation mode

[0028] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be construed as limiting the protection scope of the present invention.

[0029] Example 1. Screening of the culture medium of strain Streptomyces alboflavus sp. NX-12

[0030] The peptide compound discovered from the fermentation product of Streptomyces alboflavus NX-12 of the present invention has the molecular formula C 28 H 37 ClN6O8 and a relative molecular weight of 620.

[0031] Strain Streptomyces alboflavus NX-12 was cultured and screened under different culture medium conditions. The tested culture media included G2, F1, F2, B1, B2, YMS, NR, MKB, iron-deficient No. 5, ISP3, GYM and blank culture medium, and the preparation methods are shown in Table 1.

[0032] Table 1: Composition and preparation method of the culture medium

[0033]

[0034]

[0035] By comparing the metabolite spectra under these culture media and combining the molecular weights of the isolated products analyzed by LC-MS, the effects of different culture conditions on the synthesis of the target product were observed. After comprehensive analysis, G2 culture medium was determined as the optimal culture medium.

[0036] Example 2. Preparation and purification of peptide compounds

[0037] 1. Fermentation of the strain

[0038] Dip a sterile cotton swab into strain Streptomyces alboflavus NX-12 and evenly coat it on the MS solid culture medium plate (agar 20 g / L, mannitol 20 g / L, soybean powder 20 g / L), and incubate it upside down in an incubator at 28 °C for 3-5 d until the spores cover the surface of the plate. Use a sterile spatula to cut about 1 cm 2The agar blocks containing bacteria were inoculated into 100 mL of G2 liquid medium (20 g / L of millet, 10 g / L of grape, 3 g / L of peptone, 4.5 g / L of sodium chloride, 1 g / L of calcium carbonate), and cultured with shaking at 28 °C and 220 rpm for 48 h to prepare the seed liquid. Then, the seed liquid was inoculated into the G2 fermentation medium at an inoculation amount of 10%, and continued to be cultured at 28 °C and 220 rpm for 5 d.

[0039] 2. Extraction of fermentation products and obtaining of extracts

[0040] The fermentation broth of the strain Streptomyces alboflavus NX-12 was collected, and 2 volumes of ethyl acetate were added, and extracted overnight at room temperature. After extraction, ethyl acetate was recovered under reduced pressure using a rotary evaporator to obtain an ethyl acetate extract, which is the crude extract of the fermentation product.

[0041] 3. Purification of peptide compounds

[0042] The obtained ethyl acetate extract was further separated and purified to prepare the target compound shown in Formula I (see Figure 1 ). Through a variety of chromatographic separation techniques, including silica gel column chromatography and high performance liquid chromatography (HPLC), the extract was separated under different solvent conditions to obtain the target compound. The specific steps are as follows:

[0043] (1) Silica gel column chromatography of ethyl acetate extract

[0044] The ethyl acetate extract was mixed evenly with a small amount of silica gel (200 - 300 mesh), and then loaded into a 100 g pre-packed silica gel normal phase column for rapid separation. The dichloromethane / methanol system was used as the mobile phase, and the following three solvent systems were used for silica gel column elution in sequence: dichloromethane, dichloromethane:methanol = 1:3, and dichloromethane:methanol = 1:1. After each fraction was collected and concentrated, it was dissolved in methanol, filtered, and then subjected to liquid phase analysis. According to the HPLC chromatographic peak characteristics, the eluents with similar compounds were combined, and finally three fractions were obtained: fraction Fr.1 - Fr.3, and the target compound was present in fraction Fr.2.

[0045] HPLC analysis conditions: Agilent HPLC was used, with a diode array detector (DAD), C18 analytical column (5 μm, 250×4.6 mm), detection wavelengths 240 nm and 360 nm, injection volume 30 μL, flow rate 1 mL / min, at room temperature. Elution system: Solution A: water (0.1% formic acid), Solution B: acetonitrile; Elution conditions: 0 - 5 min, B%: 30%; 5 - 10 min, B%: 30% - 40%; 10 - 13 min, B%: 40% - 52%; 13 - 35 min, B%: 52% - 65%; 35 - 40 min, B%: 65% - 100%).

[0046] (2) HPLC purification

[0047] Solvents: Solution A: water, Solution B: methanol.

[0048] Chromatographic column: C18 chromatographic column, size 10 mm×250 mm;

[0049] Equipment: Agilent 1260 Infinity LC system, UV detector, detection wavelength range 190 - 600 nm, on - line detection wavelength 220 nm. The elution system is water (Solution A) and methanol (Solution B), elution conditions: B%: 68%, flow rate 2 mL / min, injection volume 100 μL. Collect the eluate with tR at 38 - 40 min, concentrate under reduced pressure to remove the organic phase and then lyophilize to obtain the target compound.

[0050] (3) Structure identification of the peptide compound zhengxinmycin shown in Formula I

[0051] The target compound zhengxinmycin is a white powdery solid, soluble in acetone, ethyl acetate, methanol, chloroform and acetonitrile, insoluble in water. It is colorless in acetonitrile, light yellow - green in methanol, and shows fluorescent yellow - green in DMSO. High - resolution electrospray ionization mass spectrometry (HRESIMS) shows a quasi - molecular ion peak m / z 621.0880 [M + H] + (see Figure 2 ), suggesting its molecular formula is C 28 H 37 ClN6O8, with an unsaturation degree of 13. The 1 1H NMR spectrum (CD3COCD3, 600 MHz) of the compound shown in Formula I is as Figure 3 shown; its 13 13C NMR spectrum (CD3COCD3, 150 MHz) is as Figure 4 shown. The UV spectrum of the peptide compound shown in Formula I shows maximum absorption peaks at 233 nm, 316 nm and 353 nm, indicating that its structure contains unsaturated structural units. The NMR data of the peptide compound are shown in Table 2.

[0052] Table 2 NMR data of the peptide compounds shown in Formula I

[0053]

[0054]

[0055] Example 3. Antibacterial activity experiment of zhengxinmycin

[0056] Weigh 1 mg of the target compound zhengxinmycin and dissolve it in DMSO to prepare a stock solution. The experimental subjects are four strains of bacteria, including two Gram-positive drug-resistant bacteria: Staphylococcus aureus (Staphylococcus aureus ATCC 25923) and Staphylococcus aureus (Staphylococcus aureus USA300), and two Gram-negative drug-resistant bacteria: Klebsiella pneumoniae (Klebsiella pneumoniae MGH78578) and Acinetobacter baumannii (Acinetobacter baumannii ATCC 19606). Vancomycin (1 μg / mL) and daptomycin (0.5 μg / mL) are selected as positive control drugs.

[0057] Dilute the active strain suspension of the four test bacteria to 1×108 CFU / mL, then add 90 μL of the bacterial solution to each well in a 96-well plate, and record its initial OD value. Prepare a stock solution concentration of the drug to be 160 μg / mL and serially dilute it step by step. After adding 10 μL of the drug dilution to each well, place the 96-well plate in an incubator at 37 °C for 18 h, and then use a microplate reader to measure the OD value of each well to evaluate the antibacterial activity. The results are shown in Table 3.

[0058] Table 3 MIC values (μg / mL) of the peptide compound zhengxinmycin, vancomycin and daptomycin

[0059]

[0060] As can be seen from Table 3, the peptide compound zhengxinmycin shown in Formula Ι of the present invention has good antibacterial activity. The minimum inhibitory concentration against Staphylococcus aureus ATCC 25923 and USA300 is 4 μg / mL, and the minimum inhibitory concentration against Klebsiella pneumoniae MGH78578 and Acinetobacter baumannii ATCC 19606 is 16 μm / L, indicating that it has good antibacterial activity against Staphylococcus aureus, while the antibacterial activity against Klebsiella pneumoniae and Acinetobacter baumannii is poor.

[0061] As can be seen from the above embodiments, the present invention provides a peptide antibacterial compound, a preparation method thereof and an application. The peptide antibacterial compound of the present invention has good antibacterial effects against Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii, and can be used to prepare drugs for treating bacterial infections.

[0062] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A peptide antibacterial compound, characterized in that: The structural formula of the antibacterial compound is shown in Formula I:

2. A pharmaceutically acceptable salt formed by the peptide compound according to claim 1.

3. The method for preparing the peptide antibacterial compound according to claim 1, characterized in that: The following steps are involved: S1. Spread the spores of Streptomyces alboflavinus NX-12 on a solid culture medium and culture at 26-28°C until the spores cover the entire surface; S2. Pick up Streptomyces alboflavinus NX-12 and place it in liquid culture medium for fermentation; S3. The fermentation product was concentrated under reduced pressure with ethyl acetate to obtain a crude extract; S4. Purifying the extract in S3 to obtain the antibacterial compound; The Streptomyces alboflavinus NX-12 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, with the deposit number being CGMCC No.33132.

4. The preparation method according to claim 3, characterized in that: The solid culture medium is MS culture medium; the liquid culture medium is G2 culture medium.

5. The preparation method according to claim 3, characterized in that: The fermentation culture conditions are: 26-30° C., 200-240 rpm, and culture for 4-6 days.

6. Use of the peptide compound according to claim 1 in the preparation of a drug for inhibiting Staphylococcus aureus.

7. A biological antibacterial agent, characterized in that: The peptide antibacterial compound according to claim 1 and / or the pharmaceutically acceptable salt according to claim 2 are used as active ingredients, and also include pharmaceutically acceptable excipients.

8. The biological antibacterial agent according to claim 7, characterized in that: The pharmaceutically acceptable excipients account for 0.1 to 99.9% of the total weight of the pharmaceutical composition by weight.

9. The biological antibacterial agent according to claim 8, characterized in that: The dosage forms of the biological antibacterial agent include tablets, capsules, liquid preparations and dry suspensions.

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